Study of Ras-Mediated Apoptosis

描述（由申请人提供）：一种有吸引力的抗癌策略范例是靶向癌基因，使肿瘤细胞对凋亡敏感。基本原理是癌蛋白 依赖其他因素来维持体内平衡或促进癌细胞存活。协调蛋白之一的破坏将引发癌细胞的凋亡危机。致癌性Ras已在约30-40%的人类癌症中发现。然而，靶向突变Ras的抑制剂已被证明在临床上无效。最近，研究的重点是寻找Ras下游或与Ras平行的信号通路，这些通路是癌细胞生存所必需的。在鉴定突变Ras的凋亡伴侣的努力中，研究表明，PKC的缺失与突变Ras一起在许多类型的肿瘤中是合成致死的。最近，我们提出了新的观察，不同的PKC亚型的损失差异敏感的细胞表达突变ras凋亡。特别是，我们发现PKC α/Akt的共抑制对于携带突变ras的癌细胞中诱导凋亡至关重要，其中Ras下游的PI 3 K/Akt功能动员两个信号通路：一个是通过上调ROS和ER应激介导的UPR来开启凋亡机制;另一个是通过激活PKCδ，导致p73磷酸化和p73/NOXA上调。在这个更新的应用程序中，我们将研究我们的新观察的分子机制，即Ras如何在失去 PKC α/PKC β，差异性地利用其下游效应通路选择性地使肿瘤细胞对凋亡敏感，而对正常细胞或周围组织具有较小或无毒性。我们将确定ROS和PKCδ/p73通路是如何被异常的Ras/PI 3 K/Akt重定向以在PKC α/Akt共抑制后激活细胞死亡程序的。因此，待检验的假设是，在共敲低PKC α/Akt后，PI 3 K/Akt是诱导Ras介导的凋亡的关键参与者，其通过扰乱氧化还原稳态以及通过激活PKCδ/p73介导的凋亡网络来实现。因此，形成了四个具体目标。目的1研究PKC α/Akt共抑制时ras或PI 3 K/Akt表达异常的细胞中ROS上调的机制。目的2：探讨ROS诱导表达ras或PI 3 K/Akt的细胞在PKC α/Akt共抑制下ER应激/UPR激活的机制。在目的3中，我们将确定p73在共敲低PKC α/Akt后表达异常ras或PI 3 K/Akt的细胞对凋亡的敏感性中的作用。在目的4中，我们将在体外和体内确定共抑制后表达异常ras或PI 3 K/Akt的细胞中的促凋亡作用。 PKC α/β我们的研究结果将揭示参与Ras突变和PKC α/Akt缺失之间的这种合成致死相互作用的分子，这将为设计更有效的药物来治疗携带异常ras或PI 3 K/Akt的人类癌症提供潜在的信息。

DESCRIPTION (provided by applicant): One attractive paradigm of anticancer strategies is to target oncogenes for sensitization of tumor cells to apoptosis. The rationale is that onco-proteins rely on other factors to maintain homeostasis or promote cancer cell survival. Disruption of one of coordinating proteins would trigger an apoptotic crisis in cancer cells. Oncogenic Ras has been discovered in about 30-40% of human cancers. However, inhibitors targeting mutated Ras have proved not to be effective clinically. Recently, the focus has been on finding signaling pathways that are downstream of or parallel with Ras, and are essential for cancer cells to survive. In the effort to identify the apoptotic partners of mutated Ras, studies showed that loss of PKC, together with mutated Ras, are synthetically lethal in many types of tumors. Recently, we made the novel observation that loss of different PKC isoforms differentially sensitized cells expressing mutated ras to apoptosis. In particular, we identified that co-suppression of PKC α/ß were critical for the induction of apoptosis in cancer cells harboring mutated ras, in which PI3K/Akt function downstream of Ras to mobilized two signaling pathways: one was via upregulating ROS and ER stress-mediated UPR for switching on the apoptotic machinery; and another one was through activating PKCδ, resulting in p73 phosphorylation and PUMA/NOXA upregulation. In this renewal application, we will investigate the molecular mechanisms of our novel observation of how Ras, upon loss of PKC α/ß, differentially utilizes its downstream effector pathways to selectively sensitize tumor cells to apoptosis, with less or no toxicity to normal cells or surrounding tissues. We will determine how ROS and PKCδ/p73 pathways are being redirected by aberrant Ras/PI3K/Akt to activate cell death program after co-suppression of PKC α/ß. Thus, the hypothesis to be tested is that upon co-knockdown of PKC α/ß, PI3K/Akt are key players in the induction of Ras-mediated apoptosis, which is via perturbing redox homeostasis as well as through activating PKCδ/p73-mediated apoptotic network. Accordingly, 4 Specific Aims are formed. Aim 1 will study the mechanisms by which ROS is upregulated in cells expressing aberrant ras or PI3K/Akt upon co- suppression of PKC α/ß. Aim 2 will determine the mechanisms by which ROS induces ER-stress/UPR activation in cells expressing aberrant ras or PI3K/Akt upon co-suppression of PKC α/ß. In Aim 3, we will determine the role of p73 in the sensitization of cells expressing aberrant ras or PI3K/Akt to apoptosis after co- knockdown of PKC α/ß. In Aim 4, we will determine the pro-apoptotic effect in cells expressing aberrant ras or PI3K/Akt in vitro and in vivo after co-suppression of PKC α/ß. The outcomes of our research will reveal molecules involved in this synthetic lethal interaction between Ras mutations and loss of PKC α/ß, which will provide the potential information for designing more effective drugs to treat human cancers harboring aberrant ras or PI3K/Akt.